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Genotoxicity of dicrotophos, an organophosphorous pesticide, assessed by different assays in vitro
Journal: Environmental Toxicology Manuscript ID: TOX-10-076.R1
Wiley - Manuscript type: Research Article Date Submitted by the
Author: n/a
Complete List of Authors: chen, ssu ching
Keywords: Dicrofotos, Ames test, Chromosome aberration, Comet assay
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Genotoxicity of dicrotophos, an organophosphorous pesticide, assessed
1with different assays in vitro
2Jong-C. Wua, Soi M. Chyeb, Ming –K Shihc, Chin-H. Chend, Hsin-L Yang e,* 3
Ssu. C. Chenf, * 4
a
Department of Applied Chemistry, Fooyin University, Kaohsiung, Taiwan, ROC 5
b
School of Medicine, Department of Human Biology, International Medical 6
University,No. 126, Jalan 19/155B, Bukit Jalil, 57000, Kuala Lumpur, Malaysia. 7
c
Department of Food and Beverage, National Kaohsiung Hospitality College 8
Sung-Ho Rd. Hsiao-Kang Kaohsiung Taiwan ROC
9 d
School of Pharmacy, Taipei Medical University, Taipei 110-31, Taiwan; Department 10
of Medical Technology, Yuanpei Institute of Science and Technology, Hsin-Chu, 8 11
Taiwan, ROC. 12
e
Institute of Nutrition, China Medical University, Taichung, Taiwan, ROC 13
f
Department of Biotechnology, National Kaohsiung Normal University, Kaohsiung, 14
Taiwan, ROC; Department of Life Science, National Central University, Chung-Li 15
city, Taoyuan county, Taiwan, ROC. 16
17
# Both authors contribute equally 18 19 20 21 22 23 24
Correspondence to Dr. Ssu Ching Chen* 25
Address: Department of Biotechnology, National Kaohsiung Normal University, 26
Kaohsiung, Taiwan, ROC 27
Department of Life Science, National Central University. Chung-Li city, 28
Taoyuan county, Taiwan, ROC. 29
. e-mail: [email protected] ; Fax: 886-7-6051353 30
Correspondence to Dr. Hsin-L Yang* 31
Address: Institute of Nutrition, China Medical University, Taichung, Taiwan, ROC 32 e-mail: [email protected] 33 Fax:: 04-22053366-7503 34 35 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1
Abstract 2
Dicrotophos is a systemic insecticide with a wide range of applications. We 3
investigated the genotoxicity of dicrotophos using the Ames test, the chromosome 4
aberration test in CHO-K1 cells, and the comet assay in the Hep G2 cells, while this 5
chemicals’ toxicity to both the cell lines was evaluated with the MTT assay. Results 6
showed that dicrotophos did not show any cytotoxicity to CHO-K1 cells, whereas it 7
was cytotoxic to HepG2 cells incubated for 24 h but not for 2h. For genotoxicity of 8
dicrotophos, a significant change in the numbers of bacterial reveratnts using 9
Salmomella typhimurium TA97a, TA98, TA100, TA102, and TA1535 as the tester 10
strains, an increase in the frequencies of chromosome aberration in CHO-K1 cells, 11
and an induced DNA damage in HepG2 cells were observed, indicating that 12
dicrotophos was genotoxic in these three performed assays. 13
From this study, we provide further evidence towards of genotoxic effects of 14 dicrotophos. 15 16 17 18 19 20 21
Keyworsd: Dicrofotos, Ames test, Chromosome aberration, Comet assay 22 23 24 25 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1. Introduction 1
Organophosphate (OP) insecticide exhibit a high level of pest control ability, and 2
have a relatively low degree of environmental toxicity (Poovala et al., 1999). Hence, 3
OPs are the most widely used insecticides worldwide (Sultatos, 1994). However, 4
accidental as well as suicidal poisoning cases related to these pesticides have 5
increased over the years (Bardin et al., 1994; Sultatos, 1994). 6
Dicrotophos is used against a variety of sucking, boring, and chewing insects of 7
crops both inside and outside the United States (Poovala et al., 1999), and has been 8
used as one of active ingredient in Bidrin®, an OP insecticide (Poovala et al., 1999). 9
Bidrin® is classified as a restricted-use pesticide due to its high toxicity to humans 10
and wildlife (Poovala et al., 1999). Some studies suggested the a long-term exposure 11
of OP insecticides in man results in the development of different cancers such as 12
non-Hodgkin’s lymphoma (Waddell et al., 2001; de Roos et al., 2003) and different 13
leukaemias (Brown et al., 1990). 14
As many carcinogens are genotoxic (Purchase, 1994), it is imperative to 15
investigate if OP insecticides can cause genotoxic in human or animal cells. Although 16
in vitro studies proved that certain OPs are genotoxic (Galloway et al., 1987; Vrhovac 17
et al., 2000; Bolognesi, 2003; Hreljac et al., 2008). The information on the genotoxic 18
properties of dicrotophos is limited and inconsistent. It was reported that dicrotophos 19
is mutagenic to Salmonella typhimurium TA 100 but not to TA 98 (Breau et al., 1985). 20
On the contrary, dicrotophos did not induce reverse mutations in E. coli WP2, CM561, 21
CM571, CM611, and WP12 (Hanna, 1975) and S. marcescens (Dean, 1972), whereas 22
E. coli strains WP2 uvrA and WP67 gave a positive mutagenicity towards dicrotophos 23
(Hanna, 1975). Dicrotophos also induced mitotic gene conversion in Saccharomyces 24
cerevisiae (Fahrig, 1973), and increased frequency of sister chromatid exchanges 25 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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(SCE) in cultured Chinese hamster ovary cells at a concentration of 0.3 mM (Nishio 1
and Uyeki, 1981). The major limitation of these studies was based on the 2
determination of the mutagenicity of dicrotophos without metabolic activation only. 3
As far as we know, some suspected mutagens after metabolic activation become more 4
mutagenic, which is critical in detecting compounds giving false negative results in 5
other in vitro mutagenic assays. 6
In this study, to confirm the genotoxicity of dicrotophos, the Ames test on 7
Salmonella typhimurium strains, the chromosome aberration on CHO-K1 cells or the 8
comet assay on HepG2 cells were performed in the absence and presence of metabolic 9
activation (S9 mix addition). The HepG2 cells were chosen because of their human 10
origin and their retained activities of xenobiotic-metabolizing enzymes, which make 11
them a better model for reflecting the processes in intact liver then other in vitro test 12
system (Knasmuller et al., 1998). Also, the comet assay has been validated in HepG2 13
cells as a reliable method for predicting genotoxic compounds (Uhl et al., 2000), and 14
was first adopted in detecting the genotoxicity of dicrotophos herein. 15
1. Materials and methods
16
2.1. Chemicals, cells and medium 17
Dicrotophos (purity > 99 %), 9-aminoacridine (9-AA), 2-nitrofluorene (2-NF), 18
sodium azide (SA), mitomycin C (MMC), 2-aminoanthracene (2-AA), 19
dimethylsulfoxide (DMSO), low melting-point agarose, normal melting-point 20
agarose, tris buffer, propidium iodide, trypsin, antibiotic solution, and 21
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained 22
from Sigma. Benzo [a] pyrene was purchased from Fluka. 23
2.2. Cell culture and treatment 24
For the analysis of chromosome aberration on CHO-K1 cells, CHO-K1 cells 25 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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(Chinese hamster ovarian cell line) were culture in HAM’s F12 medium (Biological 1
Industry) with 10 % of heat-inactivated fetal bovine serum (FBS) (Biological 2
Industry), 2mM L-glutamate (Biological Industry), and 100 U/mL of penicillin and 3
streptomycin (Biological Industry). The culture was incubated at 37ºC in a humid 4
atmosphere of 5 % CO2. The cells were treated with 1.5 mM, 0.75 mM and 0.375 mM 5
dicrotophos in the absence or presence of 1 % (v/v) of Aroclor-induced S9 mixture 6
(Moltox), a rat liver homogenate, for 3 h and 18 h, respectively 7
For the comet assay, HepG2 cells were grown in DMEM medium supplemented 8
with 10 % (v/v) fetal bovine serum, 2 mM L-glutamine, and 50 U/ml penicillin, and 9
0.1 mg/ml streptomycin. The cells were grown in 25-cm2 flasks at 37°C in a 10
humidified atmosphere of 5 % CO2. Subsequently, the cells were treated with 11
different concentrations of dicrotophos (0, 25, 50, 100, 200 and 400 µM) for 2 h in a 12
dark incubator. Finally, the cells were collected by centrifugation (250 g for 5 min at 13
4°C). A single cell suspension of 3 × 105 cells/mL was prepared in DMEM medium 14
without any supplement for comet assay. 15
16
2.3 Cytotoxicity assay 17
For the cytotoxicity assay, HepG2 cells or CHO-K1 cells were seeded onto 96-well 18
plates at a density of 104 cells/well and then were incubated for 24 h at 37°C to attach. 19
The medium was then replaced with fresh complete medium containing dicrotophos at 20
the indicated doses, and subsequently were incubated for 3 h and 18 h in CHO-K1 21
cells, and for 2h and 24 h in HepG2 cells, respectively, MTT (final concentration 0.5 22
mg/mL) was then added and the plates were incubated for additional 3 h. At the end 23
of incubation with MTT the medium was removed and the formazon crystals were 24
dissolved in DMSO. The optical density (OD) was measured at 570 nm (reference 25 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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filter 690 nm) using a microplate reading spectroflurimeter. Viability was determined 1
by comparing the OD of the wells containing the dicrotophos –treated cells with those 2
of the untreated cells. The results are expressed as the mean values of at least three 3
independent experiments. Student’s t-test was used for the evaluation of the statistical 4
significance between exposed and control cells; p<0.05 was considered significant. 5
2.4. Ames test 6
Mutagenicity was assessed by the preincubation assay as described by Maron and 7
Ames (1983). Briefly, 0.1 mL of overnight-grown (1-2 × 109 cfu/mL) of Salmonella 8
typhimurium strains TA 97a, TA98, TA100, TA102, and TA1535 were treated 9
separately for 30 min at 37 °C with 0.5, 5, 50,500, and 5000 μg/plate of dicrotophos, 10
both in the absence and the presence of a rat liver homogenate (S9) (0.75 %). For 11
the mutagenicity assay, the controls and dicrotophos -treated cells with mixed with 3 12
mL of sterile top agar (0.6 % agar and 0.5 %NaCl containing 0.5 mM histidine and 13
0.5 mM biotin) and poured onto minimal glucose agar plate [1× Vogel-Blonner salts 14
(0.2 g/L magnesium sulfate, 2 g/L citric acid monohydrate, 10 g/L dipotassium 15
hydrogen phosphate, and 3.5 g/L sodium ammonium phosphate), 2 % glucose, and 1.5 16
% agar]. The plates were then incubated at 37 °C for 48 h, and then the number of 17
revertant colonies was counted. Two independent experiments were conducted; each 18
experiment consisted of three replicate plates for each treatment. For the mutagenicity 19
assessment, induced responses were considered marginally positive when they were > 20
or = two-fold background colonies and positive when they were > or = three-fold 21
background colonies as described in our previous study (Wu et al., 2009). 22
2.5. Analysis of chromosome aberrations (CAs) 23
The CA assay was developed according to procedures described in OECD 24 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Guideline for testing of chemicals, # 473 (1997), The exponentially growing CHO-K1 1
cells treated with different concentrations of dicrotophos as indicated above, negative 2
control (culture medium), and positive control (0.5 µg/ mL mitomycin C and 25µg/ 3
mL benzo(a)pyrene in the absence and presence of S9 mix, respectively) for 3 h or 18 4
h. Subsequently, 10 g/ mL of colcemid (Gibco) was added for 2h. After harvest, the 5
cells were hypotonized in 0.075 M KCl solution at 37 °C for 5 min, and then were 6
fixed with a mixture of methanol/acetic acid (3:1). Finally, cells were dropped on 7
clean slides and stained with 5 % Giemsa for 10 min. A total of 200 metaphases were 8
analyzed conducted under optical microscope. The structural chromosome aberrations 9
including chromosome breakage (csb) and exchange (cse), chromatid breakage (ctb) 10
and exchange (cte), and other abnormalities were recorded, separately. Two separate 11
experiments were carried out for each treatment. Statistical analyses of these data 12
were performed using Poisson distribution. The p value less than 0.05 were 13
considered statistical significance. 14
2.6. Comet assay 15
The comet assay was performed under alkaline conditions following the method 16
of previous study (Chen et al., 2008). Ten microliters of cell suspension (2.5×105 17
cells/mL) was gently mixed with 75µL of 0.5% (w/v) of low melting point agarose 18
(LMPA). Seventy-five micro liters of this suspension was rapidly layered onto the 19
slides pre-coated with the mixtures of 0.5% normal melting pointing agarose (NMPA) 20
and 0.5% LMPA. The slides were immersed in a freshly made lysis solution (2.5M 21
NaCl, 100mM Na2EDTA, 10mM Tris and 1% (v/v) Triton X-100 at pH 10)at 4◦C for 22
1 h, and then were placed in a horizontal electrophoresis tank containing 0.3M NaOH 23
and 1mM Na2EDTA for 10 min. Thereafter, the electrophoresis (1 V/cm, 300 mA) 24 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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was conducted for 15 min at 4◦C. Subsequently, the slides were then soaked in a cold 1
neutralizing buffer (400mM Tris buffer, pH 7.5) at 4◦C for 10 min, were dried in 2
100% methanol for 5 min, and were stained with 40 µL propidium iodide (PI) (2.5 3
µg/mL). The slides were immediately evaluated at 400 × magnification using 4
fluorescence microscope (Nickon Eclipse E200). At least 100 randomly selected 5
images were analyzed from each sample and the DNA damage was analyzed with 6
Comet Assay IV software. The tail moment comet parameter (mean ± SD) measure 7
was used as DNA damage indicators in our study, since it is considered the most 8
informative and reliable measurement (Tavares et al., 2009). The results are expressed 9
as the mean values of at least three independent experiments. Student’s t-test was used 10
for the evaluation of the statistical significance between exposed and control cells; 11
p<0.05 was considered significant. 12
2. Results and discussions
13
3.1 Ames test 14
The results of the Ames test (with/without exogenous metabolic activation) 15
performed with dicrotophos were presented in Table 1. According to the historical 16
values in the laboratory, a compound tested with the Ames test was considered 17
mutagenic if the number of His+ revertant colonies was at least twice the value of the 18
corresponding solvent control (Severin et al., 2010). Dicrotophos was not cytotoxic 19
for the bacteria at any concentration with or without exogenous activation system, 20
which was based on the observations of bacterial lawn grown in the minimal glucose 21
agar plate by microscope. In addition, this chemical was only mutagenic towards S. 22
typhimurium TA 97a, TA98, TA100, TA102, and TA1535 at the highest 23
concentration (5000 µg/plate) irrespective of metabolic activation. Therefore, the 24 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Ames test did show genotoxic potential of dicrotophos compared to the respectively 1
positive control. 2
Our results for detecting the mutagenicity of dicrotophos with the Ames test 3
were contradictory to those in one paper by Breau et al. (1985) who reported that 4
dicrotophos is mutagenic to S. typhimurium TA 100 but not to TA 98 in the absence of 5
metabolic activation. In this study, the potential mutagenicity of dicrotophos was 6
assessed with the Ames test performed according to the OECD guideline no. 471. 7
Five different strains were used, in contrast to the previous study using only two 8
strains without metabolic activation. Based on the mutagenicity of dicrotophos, we 9
suggested that it induces the frameshift mutation in TA97a and TA98. Also, it induces 10
the point mutation in TA100 and TA1535 with and without metabolic activation. 11
Although TA 100 is more sensitive TA1535 for mutagen detection, 36 out of 659 12
chemicals judged as mutagens in the Ames test when subjected to the National 13
Toxicology Program’s screening protocol were evaluated as positive in TA1535 but 14
nit in TA100 (Prival and Zeiger, 1998). Thus, TA1535 was still kept as a part of the 15
core battery strains in the Ames test in present study. In addition, it also induces the 16
oxidative stress in TA102 as it can detect a variety of oxidative mutagens (Levin, et 17
al., 1982). The role of oxidative stress in Birdin®, an OP insecticide formulation with 18
dicrotophos as the active ingredient, -induced toxicity in the renal tubular epithelial 19
cell has been reported (Poovala et al., 1999). 20
21
3.2 Cytotoxicity assay 22
Nishio and Uyeki (1981) proved that dicrotophos induces statistically significant 23
increase in sister chromatid exchange in CHO cells at concentrations between 0.03 24
mM and 1 mM. Thus, dicrotophos at the similar doses ranged from 0.094 mM to 1. 5 25 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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mM were tested for its cytotoxicity and genotoxicity to CHO-K1 cells with the 1
analysis of MTT and chromosomal aberration, respectively. 2
The MTT assay demonstrated the effect of dicrotophos on the viability of 3
CHO-K1 cells (Table 2a). Results show that a number of cells with the treatment of 4
dicrotophos for 24h were increased, after metabolic activation at the tested doses over 5
0.375 mM. Without metabolic activation, the cells after dicrotophos treatment at 6
0.188 mM and 0.375 mM for 3h were also increased in their numbers compared with 7
the control group (p<0.05). However, dicrotophos at the doses of 12.5 µM to 200µM 8
was slightly cytotoxic to Hep G2 cells incubated for 24 h but not for 2 h using the 9
MTT assay (Table 2b). It seems that HepG2 cells are more sensitive to dicrotophos 10
toxicity than CHO-K1 cells. Dicrotophos has also been reported to cause cytotoxicity 11
in renal tubular epithelial cells (Poovala et al., 1999), in chick sympathetic ganglia 12
cells (Oberteiner and Sharma, 1978), and in neuroblastoma cells (Harvey and Sharma, 13
1980). 14
3.3. Chromosome aberration (CAs) 15
Dicrotophos was evaluated for its potential genotoxicity through the chromosome 16
aberration test in CHO-K1 cells. The results obtained in this assay were summarized 17
in Table 3 and Table 4. The CAs was measured by mainly counting the number of 18
metaphases with chromatid and chromosome aberration types including chromosomal 19
exchange and breaks. The other chromosomal abnormalities such as gaps, fragments, 20
ring, and dicentric were scored into one group separately. The differences observed in 21
CA frequency between the control cells and dicrotophos (0.375 mM, 0.75 mM and1.5 22
mM) -treated cells for 3 h or 18 h in the presence and absence of metabolic activation 23
were statistically significant (p < 0.01) (Table 5). Among the types of chromosome 24
and chromatid-type aberration induced by dicrotophos, chromosomal exchange and 25 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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breaks were the most evident (Table 3 and Table 4). The positive control induced a 1
significant number of cells with chromosomal aberration compared with the negative 2
control (p < 0.01). 3
As dicrotophos did not exhibit any cytotoxic effects on the growth of CHO-K1 4
cells (Table 2), and did induce a statistically significant increment in the CAs level at 5
the indicated doses irrespective of metabolic activation, we concluded that the 6
clastogenic effect observed with dicrotophos is not due to cytotoxicity. Although we 7
have not found any report of the CA test for dicrotophos, the chromosome aberration 8
assay in cultured cells has been used for many years, and it has proved to a great 9
method in the prediction of cancer risk, since there is a connection between the level 10
of chromosome aberrations and some types of cancer (Boffetta et al., 2007). 11
12
3.4. Comet assay 13
Dicrotophos was first evaluated for its potential genotoxicity in the comet assay. 14
After 2h and 24h exposure of HepG2 cells to dicrotophos nucleoid images from each 15
treatment were recorded and the values of tail moment (TM) were obtained. Table 6 16
presented the level of DNA damage in HepG2 cells., Although the viability of cells 17
treated with 12.5µM-200 µM dicrotophos for 24 h was about 78 % to 86 % (Table 18
2b), the genotoxicity of this chemicalcould still be detected if a cutoff value of 75% 19
viability (Hendersonet al., 1998) is used as the criterion of the positive response; 20
otherwise, it could produce the false positive response in the comet assay. Results 21
revealed that the TM scores increased at its concentrations from 0 µM to 400 µM. 22
Except for the cells-treated with dicrotophos at 25µM for 24h, the treated cell showed 23
a significant genotoxicity compared with the negative control group (without 24
dicrotophos addition) (p < 0.05). H2O2 as the positive control induced a significant 25 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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DNA damage when compared to the negative control group (p < 0.05). 1
Except for the HepG2 cell lines used for determination of dicrotophos 2
genotoxicity, this cell line as the detected target in the comet assay was also 3
previously used by us to evaluate the genotoxicity of some mutagens such as 4
2-aminobiphenyl and 4-aminobiphenyl (Wang et al., 2006) as well as five nitriles 5
(acetonitrile, propionitrile, methacrylonitrile, butyronitrile, and benzonitrile) (Wu et 6
al., 2009). Although the comet assay has been reported to detect the genotoxins with 7
great sensitive and to be used widely in vitro and in vivo conditions to identify 8
potentially environmental genotoxins (Moller, 2006), we first evaluated the 9
genotoxicity of dicrotophos with the comet assay. It is not possible to discuss the 10
potential carcinogenicity of dicrotophos because of the scarce information regarding 11
the link between the genotoxicity and the carcinogenicity-induced dicrotophos. A lack 12
of correlation of correlation between the genotoxicity and the carcinogenicity in some 13
pesticides including dimethoate, chlorothalonil and acetchlor was reported. (Rakitsky 14
et al., 2000). To data, no information can support for the carcinogenicity of 15
dicrotophos. Further study aiming at the evaluation of the potential carcinogenicity of 16 dicrotophos is necessary. 17 18 4. Conclusion 19
The genotoxicity of dicrotophos has been confirmed by three different in vitro 20
assays (the Ames test, the chromosome aberration, and the comet assay). Since many 21
pesticides give positive results in some tests for genotoxicity but these results are 22
frequently controversial not readily reproducible, or obtained only at the toxic dose 23
levels (Rakitsky et al., 2000). Thus, ongoing risk assessment of dicrotophos through a 24
set of short-term tests with different end-points is of importance as was stated by 25 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Manas et al. (2009) who used the comet assay and cytogenetic tests for determination 1
of genotoxicity of AMPA, the major environmental breakdown product of glyphosate. 2
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aSterile water was used as the solvent of 9-AA, SA and MMC, while DMSO was used as the solvent of 2-NF, 2-AA and B(a)P. Both sterile water
a
The tested conditions of positive controls with and without metabolic activation were listed in the below table.
Sample (Mean ± S.D.)
TA97a TA98 TA100 TA102 TA1535
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Negative control 116 ± 2 106 ± 10 17± 4 12 ± 1 102 ± 6 98 ± 8 160 ± 15 185 ± 5 11 ± 1 18 ± 3 Dicrotophos (µg/plate) 5000 235 ± 7b 227 ±10b 35 ± 10 b 50 ± 13 b 375 ± 18 b 343 ± 8 b 359 ± 6 b 382 ± 8 b 97 ± 8 b 164 ± 23 b 500 129 ± 7 130 ± 12 25 ± 12 13 ± 2 157 ± 9 114 ± 10 162 ± 7 170 ± 8 21 ± 1 20 ± 4 50 122 ± 8 113 ± 6 15 ± 4 11 ± 0 112 ± 7 88 ± 10 160 ± 5 181 ± 6 13 ± 4 17 ± 2 5 125 ± 3 107 ± 2 21 ± 1 12 ± 2 110 ± 8 89 ± 5 156 ± 7 189 ± 5 15 ± 5 19 ± 5 0.5 101 ± 10 109 ± 10 22 ± 4 13 ± 3 103 ± 7 99 ± 12 152 ± 6 177 ± 3 16 ± 4 16 ± 2 Table 1
Ames test with the strains TA7a, TA98, TA100, TA102 and TA1535 exposed to five concentrations of dicrotophos with or without exogenous activation system
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Sterile water was used as the solvent of 9-AA, SA and MMC, while DMSO was used as the solvent of 2-NF, 2-AA and B(a)P. Both sterile water and DMSO were tested as the negative control for their mutagenicities, respectively.
b
p < 0.05
Abbreviation: 9-aminoacridine (9-AA); 2-nitrofluorene (2-NF); sodium azide (SA); 2-aminoanthracene (2-AA); Benzo[a]pyrene (BP);
Mitomycin C (MMC).
TA102 mitomycin C (MMC) 0.2
TA1535 sodium azide (SA) 1
TA97a 2-aminoanthracene (2-AA) 5
TA98 2-aminoanthracene (2-AA) 1
TA100 benzo(a)pyrene (B(a)P) 1
TA102 2-aminoanthracene (2-AA) 5
TA1535
with
2-aminoanthracene (2-AA) 5
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Table 2a
The cytotoxicity of CHO-K1 cells-treated with different doses of dicrotophos in the presence and absence of S9 mix
a
P<0.05
Table 2b
The cytotoxicity in Hep G2 cells treated with different doses of dicrotophos
Treatment Viability(%) Dicrofotos Dose Short-term (2 hrs Treatment) Long-term (24 hrs Treatment) 400µM 95.6±6.2 ND a 200µM 101.6±5.9 78.9±2.9 *, b 100µM 102.0±6.7 81.7±2.3 * 50µM 105.9±5.4 80.1±3.4 * 25µM 105.6±13.4 80.1±7.1 * 12.5µM 104.4±20.2 85.6±1.0* Cell viability (%) Dicrotophos Short-term (3 hrs Treatment) Long-term (18 hrs Treatment) Dose - S9 + S9 - S9 +S9 1.5mM 99.84 ± 8.84 105.53 ± 10.19 107.47 ± 1.13 a 130.54 ± 8.80a 0.75mM 97.78 ± 5.91 102.35 ± 8.24 97.95 ± 3.04 116.00 ± 0.60 a 0.375mM 102.03 ± 2.19 114.20 ± 3.17a 96.21 ± 1.31 112.64 ± 2.94 a 0.188mM 101.80 ± 4.52 103.83 ± 1.05a 103.85 ± 4.13 99.14 ± 5.51 0.094mM 99.65 ± 2.81 96.70 ± 6.77 97.87 ± 8.54 100.60 ± 4.67 Negative (culture medium) 100.00 ± 4.20 100.00 ± 3.25 100.00 ± 1.50 100.00 ± 1.35 Mitomycin C 101.09 ± 0.96 -- 60.78 ±1.19a -- Benzo(a)pyrene -- 90.83 ±1.06a -- 63.94 ±3.15a 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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0.0µM 100.0±1.6 100.0±2.0
a
ND means “not done”. b
The “*” represents the statistical significance (P<0.05)
Table 3
The chromosomal aberrations in CHO-K1 cells treated with different dose of dicrotophos in the absence of metabolic activation for 3h.
Number of cells showing structural chromosome aberration Treatment
period (Hrs) S9
mix Groups Dosages Number of cells observed
ctba cte csb cse Other
Total number of aberrations Total cell number with aberrations 100 0 0 3 0 0 3 3 100 0 0 1 1 0 2 2 - Negative Control NA Sub-total 0 0 4 1 0 5 5 100 10 1 12 2 0 25 21 100 8 3 19 2 0 32 22 - Positive Control (MitomycinC) 0.5 µg/ml Sub-total 18 4 31 4 0 57 43 100 5 13 24 3 0 45 35 100 6 9 13 1 0 29 24 3 Hrs - Dicrotrophos 1.5 mM 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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100 5 13 13 2 0 33 26 100 4 8 9 3 0 24 16 0.75 mM Sub-total 9 21 22 5 0 57 42 a 100 4 16 12 1 0 33 24 100 4 12 12 3 0 31 24 0.375 mM Sub-total 8 28 24 4 0 64 48 100 0 0 0 0 0 0 0 100 1 0 0 1 0 2 2 + Negative Control NA Sub-total 1 0 0 1 0 2 2 100 11 1 9 2 0 23 20 100 15 2 21 1 0 39 30 + Positive Control (Benzo(a)pyrene) 25 µg/ml Sub-total 26 3 30 3 0 62 50 100 4 6 17 4 0 31 24 100 3 7 11 10 0 31 26 1.5 mM Sub-total 7 13 28 14 0 62 50 100 9 5 17 2 0 33 28 100 14 12 15 4 0 45 33 0.75 mM Sub-total 23 17 32 6 0 78 61 100 5 15 12 5 0 37 31 100 5 10 15 4 0 34 26 + Dicrotophos 0.375 mM Sub-total 10 25 27 9 0 71 57 a\ aAbbreviation: ctb-chromatid breakage; cte-chromatid exchange; csb-chromosome breakage; cse-chromosome exchange; other-other abnormalities.
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Table 4
The chromosomal aberrations in CHO-K1 cells treated with different dose of dicrotophos in the absence of metabolic activation for 18h.
Number of cells showing structural chromosome aberrationa Treatment
period (Hours) S9
mix Groups Dosages Number of cells observed
ctb cte csb cse Other
Total number of aberrations Total cell number with aberrations 100 1 0 1 0 0 2 2 100 0 0 0 1 0 1 1 － Negative Control NA Sub-total 1 0 1 1 0 3 3 100 20 3 26 5 0 54 33 － Positive Control (MitomycinC) 0.5 µg/ml Sub-total 20 3 26 5 0 54 33 a 100 2 8 16 5 1 32 24 100 5 10 22 0 1 38 32 1.5 mM Sub-total 7 18 38 5 2 70 56 a 100 11 7 17 6 0 41 28 100 6 10 6 4 2 28 26 0.75 mM Sub-total 17 17 23 10 2 69 54 a 100 4 11 9 11 3 38 20 100 6 8 8 7 2 31 20 18 Hrs － Dicrotrophos 0.375 mM Sub-total 10 19 17 18 5 69 40 a 100 0 0 2 0 0 2 2 100 0 0 1 0 0 1 1 + Negative Control NA Sub-total 0 0 3 0 0 3 3 80 39 2 52 5 0 98 58 + Positive Control (Benzo(a)pyrene ) 25 µg/ml Sub-total 39 2 52 5 0 98 58 a 100 15 4 18 6 2 45 30 + Dicrotrophos 1.5 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Sub-total 29 6 32 12 3 82 49 a 100 13 15 26 2 1 57 39 100 14 6 11 2 2 35 25 0.75 mM Sub-total 27 21 37 4 3 92 64 a 100 10 5 13 3 2 33 28 100 18 5 11 3 3 40 31 0.375 mM Sub-total 28 10 24 6 5 73 59 a Table 5The induction of chromosome aberration in CHO-K1 cells-treated with different doses of dicrotophosos in the presence and absence of S9 mix
Treatment
period S9
Mixture Test article Dosage
Aberration Frequency a A number of chromosome aberration Negative control - 5 5 1.5mM 59*,b 74 0.75mM 42* 57 Dicrotophos 0.375mM 48* 64 - S9 Mitomycin C 0.5 µg/mL 43* 57 Negative control - 2 2 1.5mM 50* 62 0.75mM 61* 78 Dicrotophos 0.375mM 57* 71 Short-term (3 hours) + S9 Benzo(a)pyrene 25 µg/mL 50* 62 Negative control - 3 3 1.5mM 56* 70 0.75mM 54* 69 Dicrotophos 0.375mM 40* 69 - S9 Mitomycin C 0.5 µg/mL 33* 54 Negative control - 3 3 1.5mM 49* 82 0.75mM 64* 92 Dicrotophos 0.375mM 59* 73 Long-term (18 hours) + S9 Benzo(a)pyrene 25 µg/mL 58* 98 Negative control - 2 2 a
The aberration frequency was presented as the number of cell with aberration in 200 metaphase cells. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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b The “ * ” represents the statistical significance (P<0.01).
Table 6
The production of DNA damage in HepG2 cells treated with different doses of dicrotophos for 2h and 24h
Treatment Dose (µM) Tail moment
(mean±SD) 2h 24h H2O2 100 16.0±1.1*,a 15.1±1.63 Dicrofotos 0 0.69±0.13 1.97±0.72 25 4.92±0.38* 2.86±1.08 50 8.21±0.46* 8.77±2.91* 100 15.9±0.54* 12.42±4.13* 200 19.0±0.59* 17.93±0.22* 400 35.0±0.93* NDb a
The “*” represents the statistical significance (P<0.01) b
.ND means” not done”.
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